Detection of MicroRNA in Apple by Stem loop RT-PCR and Different Expression of Them between in Vitro Plants and in Vivo Plants

doi:10.7668/hbnxb.2010.01.020

Abstract

Abstract: MicroRNA(miRNA) play an important role in plant growth and development.Using total RNA extracted by modified CTAB method as template,miRNA were detected by qualitative RT-PCR with stem-loop RT primers in young leave,mature leave,tender stem,young fruit and root of Hanfu apple.Fourteen kinds of miRNA sequences were identified in leaves of Hanfu apple.Semi-quantitative RT-PCR was used to investigate the expression difference of ten miRNA between in vitro plants and in vivo plants of Hanfu apple.It was found that the expressions of miR156 and miR157 were obviously upregulated in the young leaves of in vitro plants.The system of analyzing expression of miRNA by stem-loop RT-PCR in apple plants laid a sound foundation for further studying the effect of miRNA in apple growth and development.

Key words: MicroRNA, Malus domestica Borkh, Expression, RT-PCR

CLC Number:

S661.1

HUANG Fei-fei,LI He,ZHANG Zhi-hong. Detection of MicroRNA in Apple by Stem loop RT-PCR and Different Expression of Them between in Vitro Plants and in Vivo Plants[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(1): 104-109. doi: 10.7668/hbnxb.2010.01.020.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2010.01.020

http://www.hbnxb.net/EN/Y2010/V25/I1/104

References

[1] Bartel D P.MicroRNAs:Genomics,biogenesis,mechanism and function[J].Cell,2004:281-297.
[2] Llave C,Kasschau K D,Rector M A,et al.Endogenous and silencing-associated small RNAs in plants[J].Plant Cell,2002,14:1605-1619.
[3] Reinhart B J,Weinstein E G,Rhoades M W,et al.MicroRNAs in plants[J].Genes Dev,2002,16(13):1616-1626.
[4] Baskerville S,Bartel D P.Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes[J].RNA,2005,11:241-247.
[5] Mallory A C,Bartel D P,Bartel B.MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes[J].Plant Cell,2005,17:1360-1375.
[6] Chuck G,Hake S.Regulation of developmental transitions[J].Curr Opin Plant Biol,2005,8:67-70.
[7] Adai A,Johnson C,Mlotshwa S,et al.Computational prediction of miRNAs in Arabidopsis thaliana[J].Genome Res,2005,15(1):78-91.
[8] Lee R C,Ambros V.An extensive class of small RNAs in Caenorhabditis elegans[J].Science,2001,294:862-864.
[9] Lauter N,Kampani A,Carlson S,et al.MicroRNA 172 down-regulates glossy15 to promote vegetative phase change in maize[J].Proc Natl Acad Sci USA,2005,102:9412-9417.
[10] Chen C F,Ridzon D A,Broomer A J,et al.Real-time quantification of microRNAs by stem-loop RT-PCR[J].Nucleic Acids Res,2005a,33:179-187.
[11] Varkonyi-Gasic E,Wu R,Wood M,et al.Protocol:a highly sensitive RT-PCR method for detection and quantification of microRNAs[J].Plant Methods,2007,3:12.
[12] Wang Q L,Li Z H.The functions of microRNAs in plants[J].Front Biosci,2007,12:3975-3982.
[13] Gleave A P,Ampomah-Dwamena C,Berthold S,et al.Identification and characterisation of primary microRNAs from apple(Malus domestica cv.Royal Gala) expressed sequence tags[J].Tree Genet Genomes,2008,4:343-358.
[14] Chang L,Zhang Z,Yang H,et al.Detection of strawberry RNA and DNA viruses by RT-PCR using total nucleic acid as a template[J].J Phytopathol,2007,155:431-436.
[15] Lu D P,Read R L,Humphreys DT,et al.PCR-based expression analysis and identification of microRNAs[J].J RNAi Gene Silencing,2005a,1(1):44-49.
[16] Elbashir S M,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2000,15:188-200.
[17] Simovic N,Wolyn D,Jelenkovic G.Sequence analysis of 18S ribosomal RNA gene in Fragaria Ananassa Duch.cultivated octoploid strawberry[J].Plant Mol Biol,1992,18(6):1217-1220.
[18] 石琰景.果树GA生物合成基因-贝壳杉烯氧化酶和20 氧化酶的克隆及表达模式的初步研究[D].泰安:山东农业大学,2002.
[19] Dostie J,Mourelatos Z,Yang M,et al.Numerous microRNPs in neuronal cells containing novel microRNAs[J].RNA,2003,9(2):180-186.
[20] Tang F C,Hajkova P,Barton S C,et al.MicroRNA expression profiling of single whole embryonic stem cells[J].Nucleic Acids Res,2006,34:9-15.
[21] Axtell M J,Bartel D P.Antiquity of microRNAs and their targets in land plants[J].Plant Cell,2005,17,1658-1673.
[22] Li Y,Li W,Jin Y X.Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa[J].Acta Biochim,Biophys Sin,2005a,37(2):75-87.
[23] Sunkar R,Zhu J K.Novel and stress-regulated microRNA and other small RNA from Arabidopsis[J].Plant Cell,2004,16(8):2001-2019.
[24] Schwab R,Palatnik J F,Riester M,et al.Specific effects of microRNAs on the plant transcriptome[J].Dev Cell,2005,8:517-527.
[25] Wu G,Poethig R S.Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3[J].Development,2006,133:3539-3547.
[26] Gandikota M,Birkenbihl R P,Hohmann S,et al.The miRNA156/157 recognition element in the 3′UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings[J].Plant J,2007,49:683-693.
[27] Brodersen P,Sakvarelidze-Achard L,Bruun-Rasmussen M,et al.Widespread translational inhibition by plant miRNAs and siRNAs[J].Science,2008,320:1185-1190.

Please choose a citation manager

Content to export